PROJECT SUMMARY Rapid impulse propagation through the atria and ventricular conduction system (VCS) is critical for normal cardiac activation and contractility. Heritable and acquired syndromes affecting conduction velocity in these tissues account for a significant burden of arrhythmic disease and are a major cause of morbidity and mortality. Unfortunately, no therapeutic options exist for improving atrial and VCS conduction due to poor understanding of the gene regulatory networks. In order to identify essential regulators of the fast conduction gene program, we made use of a previous observation that Neuregulin-1 (NRG1) is the key mediator of VCS specification. Using a signal transduction and VCS transcriptional profiling screen, we discovered that NRG1 mediates fast conduction in the heart through the Ras-MAPK-RSK/MSK signaling pathway and the transcription factor ETV1. ETV1 is a member of the Pea3 group of E-twenty-six (ets) family transcription factors that regulates specification and electrophysiological modulation of neuronal cell types. We now present data demonstrating a critical role of ETV1 in establishing and maintaining the fast conduction gene program in the heart. ETV1 is highly expressed in the atrial pectinated myocardium and VCS, where it up-regulates the expression of key cardiac conduction genes, Nkx2-5, Scn5a (Nav1.5), and Gja5 (Cx40). Consequently, Etv1 KO mice exhibit conduction slowing in the atria and VCS with a subset displaying frank bundle branch block. Patch clamp experiments demonstrated that the normal biophysical differences in the sodium current between atrial, VCS, and ventricular myocytes were lost in Etv1 KO mice, suggesting that ETV1 regulates additional modifiers of the cardiac sodium current beyond Scn5a. Etv1 KO mice also displayed VCS hypoplasia with a proportion showing absence of the right bundle branch (RBB), mirroring the defects seen in NKX2-5 haploinsufficient mice and patients. Analysis of the NKX2-5 promoter identified a highly conserved ets-binding cluster that upon deletion using CRISPR-Cas9 in vivo recapitulated failure of RBB formation. We performed PheWAS analysis and found an association between an ETV1 sequence variant and bundle branch blocks in humans. To further explore the role of ETV1 as a transcriptional regulator of the fast conduction phenotype, we propose the following specific aims: i) determine the mechanism by which ETV1 regulates the unique biophysical properties of the sodium current in atrial and Purkinje myocytes, ii) study the functional role of ETV1 in cardiac conduction and arrhythmogenesis in the adult heart, and iii) elucidate the molecular basis of bundle branch block in carriers of the ETV1 sequence variant and in Etv1 KO mice.